Presepsin production in monocyte/macrophage-mediated phagocytosis of neutrophil extracellular traps

Presepsin, a biomarker discovered in Japan, has been clinically applied as a diagnostic aid for sepsis. Recently, however, it has been reported that presepsin levels are elevated in patients with severe systemic lupus erythematosus without infection, suggesting the existence of a production mechanism that does not involve bacterial phagocytosis. In this study, we aimed to elucidate the mechanism of presepsin production without bacterial phagocytosis and explore the clinical significance of presepsin. Neutrophil extracellular traps (NETs) were induced by Escherichia coli and phorbol myristate acetate (PMA) in neutrophils isolated from the peripheral blood of healthy subjects. NET induction alone did not increase presepsin levels, but co-culturing with monocytes significantly increased them. The addition of a NET formation inhibitor also suppressed presepsin levels, suggesting that presepsin production is greatly influenced by monocyte phagocytosis of NETs. Phagocytosis of NETs by THP-1 and U937 cells, which was induced by CD14 expression, also increased presepsin levels. This study suggests that presepsin can be used to assess the severity of inflammatory diseases, such as autoimmune diseases, and monitor treatment effects.


Results
NETs are induced by bacterial stimulation in a concentration-dependent manner. In the induction of NETs by DH5α stimulation of peripheral blood isolated neutrophils, both the ratios of Cit-H3 and SYTOX Green were not significantly different from untreated neutrophils at OD 0.01 and 0.1. However, at OD 1.0, the Cit-H3 NET ratio was 8.7 ± 1.8% (Untreated 2.9 ± 0.7%) and the SYTOX Green NET ratio was significantly increased to 12.1 ± 2.1% (Untreated 2.4 ± 0.6%) (p < 0.01) (Fig. 1A,B).
NETs express high levels of extracellular CD14, MPO, and NE. The Median fluorescence intensity (MFI) values of CD14, MPO, and NE on the cell surface by flow cytometry were 0.7 ± 0.0%, 0.3 ± 0.0%, and 1.7 ± 0.5% for untreated neutrophils and 1.6 ± 0.2%, 2.3 ± 0.6%, 6.0 ± 0.6% for neutrophils stimulated by DH5α, while the NET area was 5.1 ± 0.9%, 13.3 ± 1.9%, 28.1 ± 3.4%, respectively. CD14, MPO, and NE were highly expressed in the NET area than in the untreated cells. Specifically, the expression of CD14, the source of presepsin, was significantly higher in the NET area than in the neutrophils ( Fig. 2A). The extravasated nuclei of these cells stained positively with SYTOX green. The Cit-H3-positive NETs were extracellularly positive for MPO and NE (Fig. 2B,C). NETs stimulated by DH5α showed a higher CD14 expression than that in the untreated neutrophils, consistent with flow cytometry measurements (Fig. 2D).
Evaluation of presepsin production in the induction of NETs. After inducing PMA-NETs, we observed the morphological images of monocytes that phagocytosed NETs (Fig. 3A). NETs and monocytes were co-cultured and stained for CD14, citrullinated histones, and presepsin and evaluated by immunofluorescence imaging. Citrullinated histones were not observed in untreated cells, but DH5α-NETs were positive for the same (Fig. 3B). In addition, monocytes phagocytosed NETs with high CD14 expression, and presepsin was produced intracellularly in monocytes (Fig. 3C). Presepsin levels in untreated neutrophils, DH5α-NETs-induced NET supernatants were compared. Compared with presepsin levels in untreated neutrophils (11.8 ± 2.4 pg/mL), those in DH5α-NETs (22.5 ± 0.8 pg/mL) and PMA-NETs (20.4 ± 3.4 pg/mL) showed a statistically significant increase but did not show a dramatic difference (Fig. 3D). The induction of NETs alone did not result in significant changes in presepsin levels. However, when monocytes were co-cultured with NETs, presepsin levels in DH5α-NETs (31.4 ± 3.4 pg/mL) and PMA-NETs (92.4 ± 3.3 pg/mL) increased substantially (Fig. 3E). Furthermore, the NET ratio increased in a concentration-dependent manner under both DH5α and PMA stimulation. Presepsin levels increased similarly as monocytes phagocytosed NETs (Fig. 3F,G).
Presepsin is produced by monocytes to phagocytose NETs. In both DH5α-NETs and PMA-NETs, extracellularly released Cit-H3 was observed, and CD14 was highly expressed. In addition, CD14 and presepsin were found in cells phagocytosed by monocytes (Fig. 3C). Monocytes phagocytose NETs and take them into the cells to produce presepsin. Presepsin levels are lowered by suppressing NETs. We examined the effect of diphenyleneiodonium chloride (DPI) on presepsin production under conditions in which NETs were suppressed. Compared to that in PMA-NETs, the NET ratio in DPI (0.1-10 μM) pretreatment before PMA stimulation was lower, and NETs were suppressed (Fig. 4A). Furthermore, when the cells were co-cultured with monocytes and phagocytosed by monocytes, presepsin levels lowered in a DPI concentration-dependent manner. In other words, presepsin levels decreased in a concentration-dependent manner when NETs were suppressed (Fig. 4B). Inhibition of NETs by 10 μM DPI suppressed both DH5α-NETs and PMA-NETs. Similarly, the presepsin levels after co-culture with monocytes also decreased, and the presepsin level reflected NET ratio (Fig. 4C,D).
Evaluation of presepsin for NET suppression using cytochalasin D and sivelestat. Presepsin levels were evaluated under conditions where the phagocytic activity of monocytes was inhibited by cytochalasin D. In addition, we evaluated presepsin levels using sivelestat, an inhibitor of neutrophil elastase, which is a proteolytic enzyme of monocytes. Presepsin levels in neutrophils co-cultured with monocytes (untreated) were 30.7 ± 7.7 pg/mL; those in PMA-NETs co-cultured with monocytes were 158.7 ± 5.8 pg/mL; and those under conditions of cytochalasin D-inhibited phagocytosis were 115.7 ± 4.0 pg/mL. Inhibiting the phagocytic activity of monocytes significantly decreased presepsin levels (p < 0.01). In addition, under conditions of neutrophil elastase inhibition by sivelestat, the presepsin levels were 121 ± 19.5 pg/mL, which were significantly lower than those in the PMA-NETs phagocytosed by monocytes (p < 0.05) (Fig. 5). www.nature.com/scientificreports/ Presepsin is produced by macrophages to phagocytose NETs that express high CD14 levels. CD14 expression in U937 and THP-1 cells showed mean fluorescence intensities (MFIs) of 1.3 and 1.5, respectively. Both cell lines showed significant CD14 expression with MFIs of 5.5 and 8.6 after 1α, 25-dihydroxyvitamin D3 (VD3) stimulation to induce differentiation into macrophages (Fig. 6A). Presepsin levels (untreated) in VD3-stimulated U937 and THP-1 cells were 16.8 ± 2.6 and 15.9 ± 1.7 pg/ mL, respectively, while those after co-culture of PMA-NETs with U937 and THP-1 cells increased to 31.2 ± 2.0 and 43.5 ± 1.6 pg/mL, respectively. In addition, the presepsin levels in both cell lines decreased after NETs were suppressed by DPI treatment and the cell lines were co-cultured (Fig. 6B). Morphological observation after 4 h of co-culture of the THP-1 cells and PMA-NETs, in which CD14 expression was induced by VD3 stimulation, showed that THP-1 cells phagocytosed NETs (Fig. 6C).

Discussion
It has been reported that when neutrophils and monocytes phagocytose infectious bacteria in blood vessels, CD14 expressed on their cell surface is taken up by the cells, degraded by neutrophil elastase, and released as presepsin into the blood 14 . However, there are reports of high presepsin levels in patients with SLE without infection, and the cause of this has not been clarified 18,19 . We focused on NETs, which are known to increase in the blood in sepsis and in SLE patients, to elucidate the production mechanism of increased presepsin even in SLE patients who do not have an infection. Hakkim et al. reported that the number of NETs in the blood was higher in patients with SLE due to less degradation of NETs than in healthy subjects 20 . In addition to the conventional mechanism of presepsin production, we hypothesize a novel mechanism of presepsin production in which monocytes/macrophages produce presepsin by phagocytosing NETs.
Ratios of Cit-H3 and SYTOX Green in NETs that were released extracellularly from DH5α-stimulated neutrophils had increased in a concentration-dependent manner. However, in the case of NET induction, the NET ratio did not increase up to a DH5α culture OD of 0.1 but increased at an OD of 1.0, suggesting that a certain amount of infection is necessary for NET induction. In septic patients, when the amount of infectious bacteria in the blood is low, the bacteria are eliminated only by the phagocytic activity of neutrophils and monocytes; however, when the amount of infectious bacteria is high, the NET defense mechanism is activated in addition to phagocytic activity 35 . The induction of NETs by DH5α at an OD of 1.0 is thought to replicate the appearance of NETs in sepsis in vitro.
NETs are formed in PMA-NETs by activating neutrophil protein kinase C (PKC) to activate NADPH oxidase 36 . NETs include suicidal NETs, which are ROS production-dependent, and vital NETs, which are non-ROS production-dependent. It has been reported that suicidal NETs release citrullinated histones into the extracellular space and die after the sterilization of bacteria 35,37 . Masuda et al. have described the fractions with high forward scatter (FS) and side scatter (SS) that appeared after stimulation with DH5α as the NET area [38][39][40] . In our experiments, post stimulation with DH5α, we also observed structures in the high FS and SS fractions that were not observed in unstimulated neutrophils (Untreated). We compared the expression levels of Cit-H3 in the structures in the high FS and SS fractions with those in unstimulated neutrophils (Untreated), and found that the structures in the high FS and SS fractions expressed significantly more Cit-H3, considering that NET formation was induced. In the NET area, CD14, MPO, and NE expressions were higher than that in DH5α-activated neutrophils, suggesting that cells in the NET area are highly capable of defending themselves by increasing the expressions CD14, which serves as a sensor for recognizing bacteria, and MPO and NE, which sterilize the bacteria.
MPO released extracellularly causes MPO cytoplasmic antibody (MPO-ANCA)-associated vasculitis, leading to a vicious cycle of inflammation [41][42][43][44] . Therefore, presepsin, which can be measured easily and rapidly, could be used as a biomarker for these diseases. In studies on septic patients and mouse models, it has been reported that CD14 expression increases in an MYD88-dependent manner 13 . In our experiments, CD14 expression in neutrophils increased after stimulation with DH5α. Interestingly, flow cytometry and immunofluorescence imaging revealed that CD14 expression was higher in the NET area Braian et al. reported that macrophages phagocytosed Mycobacterium tuberculosis-induced NETs 45 . Similarly, our immunofluorescence imaging using a presepsin antibody showed that monocytes phagocytosed NETs showing high CD14 levels in both DH5α-and PMA-stimulated NETs, demonstrating the production of presepsin in monocytes. However, compared to the isolated neutrophils, no significant change in presepsin levels was observed after induction of NETs by DH5α or PMA, suggesting that NETs do not produce presepsin; rather, monocytes/macrophages produce presepsin by phagocytosis of NETs. In particular, PMA-NETs only induced NETs in the absence of bacteria, which were phagocytosed by the monocytes, resulting in an increase in presepsin levels. These results indicate that monocytes phagocytose NETs that express high CD14 levels and produce presepsin in monocytes. In addition, when the NET number was increased in a PMA concentration-dependent manner and monocytes were phagocytosed, the presepsin level also increased as the NET ratio increased, demonstrating that the number of NETs had a significant effect on presepsin production. The induction of PMA-NETs was inhibited by DPI, an inhibitor of NADPH oxidase, and the presepsin levels decreased when the cells were co-cultured with monocytes. This result demonstrated that monocytes phagocytose NETs and thus produce presepsin and the number of NETs phagocytosed directly reflects the amount of presepsin produced under the same conditions in monocytes. Similar results were obtained for DH5α-NETs.
To prove that NETs released extracellularly by neutrophils were phagocytosed by monocytes in co-cultures , we performed a phagocytosis inhibition test using cytochalasin D, which inhibits the actin polymerization of phagocytes. Presepsin production was inhibited when NETs were co-cultured with monocytes. This may be because monocytes cannot phagocytose NETs. In addition, when sivelestat, an inhibitor of neutrophil elastase, was added before co-culturing NETs with monocytes, presepsin production was suppressed. These results   We hypothesized that macrophages in tissues also phagocytose NETs and produce presepsin, as the stagnation time of monocytes in peripheral blood is several hours in vivo. Presepsin levels decreased after inhibition of PMA-NETs using DPI in PMA-NET co-culture with macrophage-induced cell lines, suggesting that macrophageinduced cell lines produce presepsin via phagocytosis of NETs. In particular, differentiated machrophage THP-1 cells actively phagocytosed PMA-NETs and showed increased presepsin levels relative to those in U937 cells. We concluded that monocytes and macrophage cell lines that phagocytose NETs were extracellularly released by neutrophils and produce presepsin.
Collectively, these results indicate that when the level of NETs increase in the blood owing to sepsis or SLE, monocytes/macrophages phagocytose NETs that express high CD14 levels on their cell surface. Thereafter, CD14 is degraded by neutrophil elastase in monocyte/macrophage cells to produce presepsin. Furthermore, in cases of increased presepsin levels in hemophagocytic syndrome, we speculated that phagocytes phagocytose CD14-expressing blood cells, resulting in increased presepsin levels (Fig. 7). The new mechanism of presepsin production is of great clinical significance because it not only explains the increased presepsin levels in SLE but also has the potential to be used as an indicator of SLE treatment efficacy. Further evidence on the mechanisms of the crosstalk between monocyte/macrophage and NETs will aid in the identification of novel therapeutic strategies for SLE and hemophagocytic syndrome.

Methods
The content and execution of the current study were approved by The Ethical Committee of the Kagawa Prefectural University of Health Sciences, Japan (No.327). All methods were carried out in accordance with the guidelines and regulations of The Ethical Committee of the Kagawa Prefectural University of Health Sciences. Written informed consent was obtained from the participants before the study.

Isolation of neutrophils.
Neutrophils were isolated at room temperature from the EDTA-anticoagulated peripheral blood of healthy volunteers by density gradient centrifugation using Polymorphprep (Cat, No.1114683; Axis-Shield, Dundee, Scotland). After centrifugation for 30 min at 500 × g, the lower cellular fraction containing neutrophils was collected, serum-free RPMI 1640 medium was added, and the neutrophils were washed by centrifugation for 10 min at 400 × g. Purified neutrophils were assayed on a Sysmex XS-800i hematology analyzer (Sysmex Corporation, Kobe, Japan), the purity of neutrophils was confirmed to be more than 98.0%.

Isolation of monocytes from PBMCs.
After density gradient centrifugation using Polymorphprep, the upper cell fraction containing peripheral blood mononuclear cells (PBMCs) was collected, serum-free RPMI 1640 medium was added, and the cells were washed twice by centrifugation for 10 min at 100 × g to remove platelets. Thereafter, monocytes were isolated from purified PBMCs using the EasySep Human Monocyte Isolation Kit (Cat. No. 19359; Stemcell Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions. Purified monocytes were assayed on a Sysmex XS-800i hematology analyzer, the purity of monocytes was confirmed to be more than 94.0%.

Evaluation of Presepsin levels by co-culturing with monocytes after NET induction. PMA-
NETs were collected by pipetting to make a neutrophil suspension containing NETs. Purified monocytes (5.0 × 10 5 cells per well) from the peripheral blood of the same subject were seeded in 24-well plates, and the Figure 7. Schematic representation of the mechanism of presepsin production by monocytes/macrophages by NETphagocytosis. Neutrophils invoke NETs in response to infectious bacteria in blood vessels, trapping bacteria and turning them into dead cells. Phagocytosis of NETs by monocytes/macrophages results in intracellular degradation of CD14, which is expressed highly in NETs, and the production of a presepsin, which is released into the extracellular space. www.nature.com/scientificreports/ ratio of purified neutrophils (control) to that of the neutrophil suspension containing NETs added was the same. After incubation for 3 h at 37 °C, presepsin in the supernatant was measured using PATHFAST (LSI Medience Corporation, Tokyo, Japan).
Immunofluorescence imaging of NETs. Purified neutrophils were seeded in 24-well plates with submerged coverslips at the bottom, and NETs were induced using DH5α. After stimulation, the medium was removed, and the remaining cells were washed with phosphate-buffered saline (PBS). Cells on the coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, cells were incubated in PBS containing 5% rat serum for 60 min to block non-specific antibody binding. The samples were then incubated for 60 min with the following primary antibodies: anti-human citrullinated histone H3 (Cit-H3) rabbit polyclonal antibody (dilution 1:1,000), anti-human MPO mouse monoclonal antibody (dilution 1:2,000) or anti-human NE mouse monoclonal antibody (dilution 1:2,000) or anti-human CD14 mouse monoclonal antibody (dilution 1:500). After washing in PBS, each primary antibody binding was visualized using secondary antibodies coupled to Alexa Fluor 405-conjugated goat anti-rabbit IgG (dilution 1:1,000) and rhodamine (TRITC)-conjugated goat anti-mouse IgG (dilution 1:2,000), and the samples were stained for DNA (Sytox Green; dilution 1:2,000). After incubation for 60 min, samples were washed with PBS and embedded in 80% glycerol. All the procedures were performed at room temperature 46,47 . Images were obtained using a confocal microscope (FLUOVIEW FV10i; Olympus Corporation, Tokyo, Japan).
Immunofluorescence imaging of monocytes that had phagocytosed NETs. Purified monocytes were seeded in 24-well plates with coverslips submerged at the bottom and co-cultured with monocytes after induction of NET formation. After incubation for 3 h at 37 °C, the medium was removed, and the remaining cells were washed with PBS. Cells on the coverslips were fixed with 4% paraformaldehyde. After washing with PBS, samples were incubated in PBS containing 5% rat serum for 60 min. Thereafter, the samples were incubated for 60 min with the following primary antibodies: anti-human cit-H3 rabbit polyclonal antibody (dilution, 1:1,000) and anti-human presepsin mouse monoclonal antibody (dilution, 1:5,000). After washing in PBS, each primary antibody binding was visualized using secondary antibodies coupled to Alexa Fluor 405-conjugated goat anti-rabbit IgG (dilution 1:1,000) and rhodamine (TRITC)-conjugated goat anti-mouse IgG (dilution 1:2,000). Subsequently, the samples were stained for DNA (Sytox Green; dilution 1:2,000) or CD14 using FITCconjugated anti-human CD14 mouse monoclonal antibody. After incubation for 60 min, samples were washed with PBS and embedded in 80% glycerol. All the procedures were performed at room temperature. Images were acquired using a confocal microscope (FLUOVIEW FV10i).
NET ratio and presepsin evaluation after inhibitor treatment. Neutrophils were treated with DPI, which inhibits NADPH oxidase activity. Prior to DH5α or PMA stimulation, purified neutrophils (1.8 × 10 6 cells/ well) were exposed to 10 μM DPI for 30 min at 37 °C. After NET inhibition, the NET ratio was analyzed using flow cytometry, and presepsin levels were measured after monocyte phagocytosis. Cytochalasin D was used as a phagocytosis inhibitor, and sivelestat was used as an neutrophil elastase inhibitor. Before phagocytosis of NETs by monocytes, purified monocytes (5.0 × 10 5 cells/well) were exposed to 50 μM cytochalasin D or 50 μM sivelestat for 30 min at 37 °C. After co-culture with NETs, the presepsin levels were measured.

NET phagocytosis by macrophages increases presepsin levels.
To differentiate U937 and THP-1 cells into active macrophage-like cells, the cells were resuspended in a culture medium containing 100 nM VD3 to a density of 2.0 × 10 5 cells/mL and incubated for 48 h at 37 °C. After differentiation, the cells were co-cultured with PMA-NETs derived from neutrophils obtained from healthy volunteers for 3 h, and presepsin in the supernatant was measured.
Statistical analyses. All statistical analyses were performed using SPSS version 24.0 (SPSS Inc, Chicago, IL, USA). The data are presented as the mean ± standard deviation (SD), and Student's t-test was used for comparisons between two groups. A p-value of less than 0.05 was considered statistically significant. In the graphically represented data, *, and ** denote p values of less than 0.05, 0.01, respectively.

Data availability
On reasonable request to the corresponding author, data supporting the findings of this study will be available after approval from the Ethical Committee of the Kagawa Prefectural University of Health Sciences.